1. Field of the Invention
This invention is in the field of biochemistry and more specifically relates to genetic toxicology.
2. Description of the Prior Art
Compounds or other agents which can chemically alter the DNA of a cell are capable of inducing genetic diseases such as Lesch-Nyhan syndrone, hemophilia, sickle cell anemia, and cystic fibrosis. Compounds or agents having this potential are known as mutagens. In addition, most mutagens also have the capability of inducing cancer in test animals. Clearly, it is desirable to have methods for determining the potential mutagenicity of such agents in a practical manner, and bacterial mutation assays have been proposed for this reason.
One of the most commonly used bacterial assays for mutagenicity is known as the Ames assay and employs a set of Salmonella typhimurium strains which are permeable to a wide range of chemicals and also are partially deficient in DNA repair. Ames, B. N., McCann, J. and Yamasaki, E. Mutation Research 31, 347-379 (1975). In this system, a chemical's mutagenicity is determined by ability to revert a set of histidine-requiring mutants of S. typhimurium back to histidine prototrophy through reverse mutation of the original DNA lesion or through a second site mutation. While the Ames assay is a valuable genetic tool, its basic design makes it undesirable for screening purposes. This is because the Ames assay is a reverse mutation assay in which a mutant DNA locus must be specifically mutated back to its wild-type configuration or suppressed. A chemical mutagen which cannot affect the required change in the DNA will go undetected. Such mutagen specificity is known, of course.
Because of the problems with reverse assays, it would be desirable to provide a forward mutation assay based upon the inability of bacterial cells to enzymatically convert a drug to a toxic metabolite. In such an assay, a large portion of a structural gene serves as the target for the chemical mutagen. Base-pair substitutions affecting amino acids at the catalytic site or at key structural positions would be observed as a phenotypic change to drug-resistance. Frameshifts occurring over most of the structural gene, except perhaps the terminal codon sequences, would similarly be expressed. With such advantages in mind, forward mutation assays based on drug-resistance have been proposed for bacterial cells.
For example, Ellenberger and Mohn have developed a forward mutation assay based on 5-methyltryptophan in E. coli. See Ellenberger, J. and Mohn, G., Arch. Toxicol. 33, 225 (1975). Forward mutations leading to 5-methyltryptophan resistance are known to occur in five different genes. This sytem has been shown to respond to a variety of mutagens. There are some serious practical problems with the assay, however. Varying the concentration of 5-methyltryptophan changes the observed mutant fraction. Also, 5-methyltryptophan-sensitive bacteria are not completely inhibited by 5-methyltryptophan and continue to grow slowly in its presence on the plate. See Mohn, G., Mut. Res. 20, 7 (1973). This complicates the quantitation of mutation.